The goal of this project is to determine the mechanisms by which viral matrix (M) proteins and envelope glycoproteins mediate envelopment of viral nucleocapsids. This is a fundamental question in assembly of all enveloped viruses that have M proteins as part of their structure. Our work has addressed this question using vesicular stomatitis virus (VSV), which has been widely used to study virus assembly. We now know that the VSV M protein and envelope glycoprotein (G protein) interact with a variety of different viral and host components during the process of virus assembly by budding. We have developed a unique combination of genetic, morphological, and biophysical assays for studying these functional interactions. Aim 1 is to determine the membrane microdomains involved in virus assembly and phenotypic mixing. We will use our newly developed methods for analysis of immunoelectron microscopy data to quantify the organization of the viral and host proteins in membrane microdomains. In Aim 2 a mutagenesis approach will be taken to identify the "protein interaction surfaces" responsible for M protein interactions with the G protein and host membrane components (Aim 2a). The effects of these mutations will be correlated with analysis of M protein conformation and with the recently determined X-ray structure of M protein fragments. Aim 2b is to determine the interactions of M protein with viral nucleocapsids that are important for recruitment of nucleocapsid-M protein complexes to the budding site. Aim 2c involves incorporation of M protein mutations into new recombinant viruses which can be used to isolate new intermediates in the assembly process by changing the rate-limiting steps in virus assembly. These experiments will provide substantial new information about the mechanisms that underlie the critical steps in virus assembly. These data will address long-standing questions about the specificity of the assembly process such as how nucleocapsids are selected for assembly and the mechanism of phenotypic mixing of viral envelope glycoproteins. In addition, they should address the more recently recognized problem of understanding the remarkable versatility of M protein in performing different functions in virus-infected cells.